The Journal of Maternal-Fetal and Neonatal Medicine, October 2010; 23(10): 12301236

Intra-amniotic infection increases amniotic lamellar body count before 34 weeks of gestation

HIROYUKI TSUDA1,2, YUICHIRO TAKAHASHI2, SHIGENORI IWAGAKI2, ICHIRO KAWABATA2, HIROMI HAYAKAWA1, TOMOMI KOTANI1, KIYOSUMI SHIBATA1, & FUMITAKA KIKKAWA1
1

Department of Obstetrics and Gynecology, Nagoya University School of Medicine, Nagoya, Japan, and 2Department of Fetal and Maternal Medicine, Nagara Medical Center, Gifu City, Japan (Received 6 November 2009; revised 14 December 2009; accepted 18 December 2009)

Abstract Objective. To examine the lamellar body count (LBC) value in intra-amniotic infection cases and evaluate its association with the incidence of respiratory distress syndrome (RDS). Methods. Three hundred sixty-ve amniotic uid (AF) samples were obtained at caesarean section from 27 to 38 weeks of gestation. LBC and glucose concentrations in AF were measured with no centrifugation. We dened AF glucose concentrations 50.8 mmol/L and positive C-reactive protein (CRP) of the neonates as intra-amniotic infection. Results. An LBC cutoff value of 29,500/mL resulted in 94.0% sensitivity, 82.4% specicity, and 99.1% negative predictive value (NPV) for RDS. Neonates with glucose concentrations 50.8 mmol/L in AF and positive CRP had no RDS and signicantly higher LBC values than controls before 34 weeks of gestation (17.0 vs. 4.3, p 5 0.05 and 25.5 vs. 5.0, p 5 0.05, respectively), but there were no signicant differences after 34 weeks of gestation. Conclusions. LBC is an accurate predictor of foetal lung maturity and our LBC cutoff value had a high NPV for predicting RDS. We showed that intra-amniotic infection was associated with signicantly higher LBC values than the value in controls before 34 weeks of gestation, which correlated with a low incidence of RDS.

Keywords: Lamellar body count, glucose concentration, foetal lung maturity, respiratory distress syndrome, intra-amniotic infection, chorioamnionitis

Introduction Respiratory distress syndrome (RDS) of the neonate is a major cause of morbidity and mortality in infants born preterm [1]. For that reason, the accurate antenatal prediction of foetal lung maturity based on results from amniotic uid (AF) samples is of utmost importance in the prevention of neonatal RDS and its complications. The lamellar body count (LBC) was rst described by Dubin in 1989 [2]. The lamellar body is a surfactant-containing lamellated structure which is secreted by the type II pneumocyte [3]. Thus, LBC is used to estimate surfactant production in utero and predict the degree of foetal lung maturity. Several studies have shown LBC to be

an accurate predictor of foetal lung maturity [46]. LBC can be performed quickly and cheaply, so it is more cost effective predictor for the occurrence of RDS compared with the lecithin/sphingomyelin (L/S) ratio [6]. Previous studies of the relation between intraamniotic infection or histological chorioamnionitis and RDS have shown varying results [79]. However, recent reports support the concept that intraamniotic infection or histological chorioamnionitis contributes to the low incidence of RDS in preterm infants [10,11]. In animal models, intra-amniotic endotoxin injection causes inammation of the chorioamnion, increases inammatory cells in the AF, increases IL (interleukin)-1 and IL-6 mRNA

Correspondence: Hiroyuki Tsuda, MD, Department of Obstetrics and Gynecology, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Tel: 81-52-744-2261. Fax: 81-52-744-2268. E-mail: hiro-t@med.nagoya-u.ac.jp ISSN 1476-7058 print/ISSN 1476-4954 online 2010 Informa UK, Ltd. DOI: 10.3109/14767051003615442

Infection increases amniotic lamellar body expression by the chorioamnion and cells in the AF, and results in enhancement of lung maturation by inducing surfactant protein synthesis [12,13]. In the present study, we investigated whether intra-amniotic infection or histological chorioamnionitis is associated with a low incidence of RDS. If intra-amniotic infection decreases the incidence of RDS, the value of LBC should be expected to increase signicantly in those neonates. Nothing has been reported about the LBC values in intraamniotic infection cases. Then, we also investigated the LBC values in intra-amniotic infection cases and compared with controls. Methods Data were collected from April 2006 to March 2009 at the Nagara Medical Center, Gifu City, Japan. Four hundred AF samples were obtained at caesarean section (CS) from 27 to 38 weeks of gestation with informed consent. Samples were analysed immediately after arrival at the laboratory with no centrifugation, according to a standardised methodology for LBC reported by Neerhof et al. [5]. In AF, we measured LBC (per microlitre), glucose concentration (millimols per litre), and culture. LBC was determined using a platelet channel on the Sysmex SF-3000 (Sysmex, Kobe, Japan). The procedure for measuring these items took less than one hour. Neonatal data including gestational age (GA) at delivery, birth body weight, Z score of birth body weight, and neonatal RDS were abstracted. Clinical characteristics of the mother such as preeclampsia, diabetes mellitus (DM) including preexisting and gestational [14], and histologic chorioamnionitis [15] were studied. Gestational age was determined by crown-rump length using an ultrasonogram at around 10 weeks of gestation. Foetal growth restriction (FGR) was dened as birth body weight less than 7 1.5 SD for gestational age in Japan [16]. The diagnosis of RDS was established by the neonatologist based on the combination of clinical signs, chest X-ray ndings, and clinical course; the neonatologist was unaware of the LBC data. Caesarean deliveries were done for standard obstetrical indications. In the present study, we dened AF glucose concentrations 50.8 mmol/L and positive C-reactive protein (CRP) (4 0.3 mg/dL) of the neonates as intra-amniotic infection. Neonatal CRP was measured immediately after birth. The term, intraamniotic infection, is especially independence of histological chorioamnionitis. The data were collected and entered on a computerised spreadsheet (Excel spreadsheet, Microsoft, Tokyo, Japan). The data were statistically

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analysed with Dr. SPSS II (SPSS Inc., Tokyo, Japan) using descriptive statistics, w2 analysis, Students t-test, multiple logistic regression, and receiveroperating characteristic (ROC) curve. A p value of less than 0.05 was considered signicant. Results At rst, we enrolled 400 neonates in which AF could be collected at caesarean section (Figure 1). We excluded 35 neonates (foetal congenital diaphragmatic hernia, 4; foetal pleural effusion, 7; foetal heart failure due to structural abnormality, 2; neonatal death, 6; delivery after 39 weeks, 16), and 365 neonates were analysed nally. Neonatal death includes one Potter syndrome, one agenesis of the mandible, one Kasabach-Merritt syndrome, and three 18 trisomy. We divided the neonates into two groups: the group before 34 weeks of gestation (n 42) and the group at 3438 weeks of gestation (n 323), because the risk for RDS is very low after 34 weeks of gestation [17]. Background and characteristics of the cases between the two groups are shown in Table I. There were 17 neonates (4.7%) suffering from RDS. LBC value was signicantly lower in the group delivered before 34 weeks of gestation than in the group delivered after 34 weeks of gestation (7.9 vs. 11.7; p 5 0.05). There were signicantly more chorioamnionitis and RDS neonates in the group delivered before 34 weeks of gestation (p 5 0.05). The rates of FGR, preeclampsia and DM were not different signicantly between the two groups. The LBC value in AF ranged from 1000 to 577,000 /mL. We analysed the cutoff value of LBC for predicting RDS using an ROC curve and established the value of 29,500 /mL. The LBC cutoff value in the present study resulted in 94.0% sensitivity, 82.4% specicity, 40.0% positive predictive value (PPV), and 99.1% negative predictive value (NPV) for predicting RDS.

Figure 1. Algorithm of inclusion and exclusion cases in this study. CS, caesarean section; CDH, congenital diaphragmatic hernia; ND, neonatal death.

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H. Tsuda et al. LBC values in each clinical situation compared with controls at 3438 weeks of gestation are shown in Table III. Neonatal RDS cases had signicantly lower LBC values than those of controls (0.5 vs. 11.8, p 5 0.05). LBC values in neonates with glucose concentrations 50.8 mmol/L in AF, CRP-positive status, and chorioamnionitis had no signicant difference compared with those of controls (p 0.141, 0.626, and 0.401, respectively). The relationship of the LBC value between intra-amniotic infection and controls is shown in Figure 2. Clinical data, LBC value, and RDS in neonates with intra-amniotic infection before 34 weeks of gestation are summarised in Table IV. Thirteen neonates met this criteria and none of them had RDS. Histologic chorioamnionitis was conrmed in ve neonates (38.5%). In case #1 and case #6, Enterococcus faecalis was identied in AF culture. The LBC value in case #2 and case #5 only were below 50,000 /mL, but case #5 was complicated by maternal DM. Since we have no cases performed caesarean section before 34 weeks of gestation without any complications, we could not compare the LBC values between intra-amniotic infection cases and normal controls before 34 weeks of gestation. Then, we compared the LBC values

LBC values in each clinical situation compared with controls before 34 weeks of gestation are shown in Table II. Neonatal RDS cases had signicantly lower LBC values than those of controls (2.3 vs. 11.1, p 5 0.01). Neonates with glucose concentrations 50.8 mmol/L in AF and CRP-positive status had signicantly higher LBC values than those of controls (17.0 vs. 4.3, p 5 0.05 and 25.5 vs. 5.0, p 5 0.05, respectively). Multivariate analysis revealed that glucose concentrations 50.8 mmol/L in AF and CRP positive status of the neonate were independently and signicantly associated with increased LBC values. Thus, the neonates with intra-amniotic infection had signicantly higher LBC values than those of controls. Clinically, Neonates with glucose concentrations 50.8 mmol/ L in AF and CRP-positive status had a signicantly lower occurrence of RDS than controls (0/11 vs. 15/ 31, p 5 0.01 and 0/6 vs. 15/36, p 5 0.05, respectively). Histologic chorioamnionitis also had signicantly higher LBC values than those of controls (15.3 vs. 5.9, p 5 0.05), but did not signicantly associate with a decrease in the occurrence of RDS (3/9 vs. 12/ 33, p 0.866). Other factors such as FGR, preeclampsia, and DM did not affect the LBC values signicantly.

Table I. Background and characteristics of the cases before 34 weeks of gestation and 3438 weeks of gestation (n 365). 534 weeks (n 42) GA at delivery, weeks (mean + SD) Neonatal body weight, g (mean + SD) Growth SD (range) Lamellar body counts, 104/mL (range) FGR Preeclampsia DM Histologic CAM UAREDF RDS 31.1 1563 70.70 7.9 8/42 6/42 3/42 9/42 5/42 15/42 (+1.9)* (+352)* (74.8 to 2.2) (0.157.7)* (19.0%) (14.3%) (7.1%) (21.4%)* (11.9%)* (35.7%)* 3438weeks (n 323) 36.9 2539 70.64 11.7 79/323 46/323 14/323 2/323 4/323 2/323 (+1.2) (+513) (75.3 to 3.6) (0.247.3) (24.5%) (14.2%) (4.3%) (0.6%) (1.2%) (0.6%)

CAM, chorioamnionitis, UAREDF, umbilical artery reversal end-diastolic ow. *p 5 0.05 compared with 3438 weeks. Table II. LBC values in each clinical situation compared with controls before 34 weeks of gestation (n 42). LBC (104/mL) (+SD) Present Glucose 50.8 mmol/L in AF CRP positive of neonate RDS PROM Histologic CAM FGR Preeclampsia DM UAREDF PROM, premature rupture of membranes. 17.0 + 16.5 25.5 + 19.5 2.3 + 3.3 8.1 + 7.6 15.3 + 17.0 7.9 + 11.0 2.1 + 1.7 1.8 + 1.5 11.0 + 13.2 (n 11) (n 6) (n 15) (n 9) (n 9) (n 8) (n 6) (n 3) (n 5) Absent 4.3 + 5.4 5.0 + 5.7 11.1 + 12.9 7.9 + 12.2 5.9 + 8.5 8.0 + 11.5 8.9 + 11.9 8.4 + 11.6 7.5 + 11.2 (n 31) (n 36) (n 27) (n 33) (n 33) (n 34) (n 36) (n 39) (n 37) p value 50.05 50.05 50.01 n.s. 50.05 n.s. n.s. n.s. n.s.

Infection increases amniotic lamellar body

Table III. LBC values in each clinical situation compared with controls at 3438 weeks of gestation (n 323). LBC (104/mL) (+SD) Present Glucose 50.8 mmol/L in AF CRP positive of neonate RDS PROM Histologic CAM FGR Preeclampsia DM UAREDF 13.3 + 8.9 (n 89) 13.6 + 7.8 (n 4) 0.5 + 0.4 (n 2) 11.8 + 6.1 (n 7) 16.4 + 7.9 (n 2) 12.8 + 8.2 (n 79) 10.2 + 8.3 (n 46) 14.5 + 9.0 (n 14) 12.8 + 9.9 (n 4) Absent 11.0 + 7.4 11.7 + 7.9 11.8 + 7.9 11.7 + 8.0 11.7 + 7.9 11.3 + 7.8 12.0 + 7.8 11.6 + 7.8 11.7 + 7.9 (n 229) (n 319) (n 321) (n 316) (n 321) (n 244) (n 277) (n 309) (n 319)

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p value n.s. n.s. 50.05 n.s. n.s. n.s. n.s. n.s. n.s.

Figure 2. The relationship of the LBCs between intra-amniotic infection and controls. IAI, intra-amniotic infection.

between intra-amniotic infection cases before 34 weeks of gestation (n 13) and repeat CS cases with no complication and no labor at term (n 100). The values of LBC in intra-amniotic infection cases before 34 weeks of gestation were higher than repeat CS cases at term, but not signicant (16.6 vs. 11.2, p 0.22). Discussion In foetal life, surfactant is stored in the cytoplasm of the alveolar type II pneumocyte in the form of lamellar bodies. They are secreted into the alveolar space and pass into the amniotic cavity and hence are found in AF. [18] Lamellar bodies are similar in size to platelets and can be counted in the platelet channel of most electronic cell counters [5], which have become widespread because they are cheap, reliable, easy to use, not time-consuming, and amenable to repetition in any institution [4]. Several studies have shown the LBC to be an accurate predictor of foetal lung maturity and established cutoffs for LBC, but caution should be exercised when interpreting data since factors such as centrifugation, freezing, and instrumentation can all affect the enumeration of lamellar bodies [18]. That is why Neerhof et al. established a consensus regarding a standardised methodology for LBC [5]. In the present study, we used the Sysmex brand of

hematology analyser, but the majority of published studies have utilised the Coulter brand. The results may differ with other brands of blood cell counters because of size interval differences in the platelet counting channel [19]. We suggest that analyserspecic cutoffs are required and should be conrmed with outcome-based studies by each laboratory offering the test. In the present study, we established the cutoff value of LBC as 29,500/mL in RDS neonates. Our results showed that the LBC cutoff value for predicting RDS had a low PPV (40.0%) but high NPV (99.1%) using the ROC curve. This suggested LBC value of greater than 29,500 /mL means reassuring ndings for RDS. The LBC cutoff value for predicting RDS in the present study is similar to that reported in the previous studies [6]. In the present study, AF samples for measuring LBC were obtained at CS cases only. So there has a limitation in the case of spontaneous birth. We dened AF glucose concentrations 50.8 mmol/L and CRP positive status of the neonates as intra-amniotic infection in the present study. For predicting intra-amniotic infection, AF culture is the gold standard [20], but results are not immediately available and may take up to 5 days. Moreover, antibiotic administration before amniocentesis might result in the failure of the positive microorganisms. The rapid diagnosis of infection is important in clinical medicine, so we used AF glucose concentrations for the detection of intra-amniotic infection. AF glucose concentrations less than 50.8 mmol/L provided a high sensitivity, specicity, and NPV for intra-amniotic infection [21]. Different from other markers such as cytokines (e.g. IL-6, 8) and leukocyte esterase, AF glucose determination is a rapid, inexpensive, and simple test and can be performed easily in most obstetric units [21]. Positive CRP of the neonates just after birth directly indicates the presence of infection or inammation during foetal life by intra-amniotic infection.

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Table IV. Clinical data, LBC value and RDS in neonates with intra-amniotic infection before 34 weeks of gestation.

Case (no.) 1 2 3 4 5 6 7 8 9 10 11 12 13

GAD (weeks) 28 29 29 30 31 32 33 33 33 33 33 33 33

FBW 1286 1718 1316 1456 1878 1818 766 1750 2562 1974 1818 1638 2174

Growth SD 0.2 2.1 70.3 70.7 0.7 70.3 74.8 71.0 2.2 70.4 71.1 71.4 0.3

Glucose 50.8 in AF 7 7

CRP () of neonate 7 7 7 7 7 7 7

LBC (104/mL) 22.6 2.2 13.1 10.6 3.4 57.7 23.1 36.9 17.6 7.8 7.7 5.5 7.2

RDS 7 7 7 7 7 7 7 7 7 7 7 7 7

Note CAM CAM CAM Maternal DM CAM

CAM

GAD, gestational age at delivery; FBW, foetal body weight.

Elevated CRP of the neonate is one of the clinical ndings of neonatal sepsis [22], but there are three cases of chorioamnionitis and two cases of positive AF culture in six cases with positive CRP of the neonate before 34 weeks of gestation. Because our cases in the present study are somewhat few, a larger study is necessary to evaluate about the correlation between neonatal CRP and infection. The majority of recent reports support the concept that intra-amniotic infection or histologic chorioamnionitis contributes to the low incidence of RDS in preterm infants [10,11]. Our results showed that neonates with intra-amniotic infection had signicantly higher LBC values than controls and had no RDS clinically before 34 weeks of gestation. This is the rst report regarding LBC values in intraamniotic infection. We speculate that intra-amniotic infection can accelerate foetal lung maturity before 34 weeks of gestation, which correlates with increased LBC value. A problem in the present study may be heterogeneity of the population. Some clinical situations such as DM can vary foetal lung maturity, so we performed multivariate analysis. Multivariate analysis revealed that intra-amniotic infection cases before 34 weeks of gestation were independently and signicantly associated with increased LBC values. Naturally, we have no cases performed caesarean section before 34 weeks of gestation without any complications, so we could not compare the LBC values between intra-amniotic infection cases and normal controls before 34 weeks of gestation. But even when we compared the LBC values between intra-amniotic infection cases before 34 weeks of gestation and repeat CS cases with no complication and no labour at term, the values of LBC in intraamniotic infection cases before 34 weeks of gestation were higher. Therefore, we speculate intra-amniotic

infection has a great impact to increase the LBC value and accelerate foetal lung maturity. At 3438 weeks of gestation, LBC values in neonates with intra-amniotic infection had no signicant difference compared with controls. Moreover, the LBC values of intra-amniotic infection cases are higher before 34 weeks of gestation than after 34 weeks of gestation. We speculate a problem with the accuracy of AF glucose concentrations for the detection of intra-amniotic infection after 34 weeks of gestation. Weiss et al. reported that mean AF glucose concentration decreases from 2.5 mmol/ L at 1417 weeks of gestation to 0.8 mmol/L at the end of pregnancy [23]. The tenth percentile of AF glucose concentration was 0.7 mmol/L at 34 weeks of gestation to 0.6 mmol/L at 39 weeks of gestation. So we speculate that some normal neonates having AF glucose concentration less than 50.8 mmol/L were included as intra-amniotic infection cases at 3438 weeks gestation. There is an overlap between intra-amniotic infection, histologic chorioamnionitis, and PROM clinically, but not equal. In the present study, histologic chorioamnionitis neonates had signicantly higher LBC values than controls but the rates of RDS were not signicantly different compared with controls before 34 weeks of gestation. We speculate that histologic chorioamnionitis without intra-amniotic infection neither increases the LBC value nor decreases the occurrence of RDS, which is similar in PROM cases. The diagnosis of intra-amniotic infection is not always conrmed by histological or microbiological studies. In one study, histological examination of the placenta did not support the clinical intra-amniotic infection in approximately one-third of cases [24]. Cultures of the AF or membranes did not document a bacterial infection in 25% to 30% of placentas that had histological

Infection increases amniotic lamellar body CAM [25,26]. Interestingly, a recent study showed that histologic chorioamnionitis without foetal involvement was associated with decreased severe RDS, whereas the effect of chorioamnionitis with foetal involvement on the incidence of severe RDS was gestational dependent [22]. Time after exposure to the infection may be the important factor regarding the occurrence of RDS. It has been reported that FGR in preterm birth can accelerate foetal lung maturity in association with prolonged intrauterine stress [27], but later this hypothesis was not supported [28]. In the present study, FGR was not signicantly associated with changed LBC values before and after 34 weeks of gestation. Also, maternal DM was not signicantly associated with changed LBC values before and after 34 weeks of gestation, but maternal DM cases in the present study were well-controlled. Piper et al. reported that neonates of diabetic women with good glycemic control have foetal lung maturation at the same gestational age as nondiabetic women [29]. In conclusion, LBC can be an accurate predictor of foetal lung maturity and our LBC cutoff value of 29,500 /mL had a high sensitivity (94.0%) and NPV (99.1%) using the ROC curve. Neonates with intraamniotic infection have signicantly higher LBC values than controls before 34 weeks of gestation, but not signicant after 34 weeks gestation. This is the rst report regarding LBC values in intra-amniotic infection, and clinical data in the present study suggest that intra-amniotic infection presented signicantly higher LBC values than controls, which correlated with a low incidence of RDS before 34 weeks of gestation. Acknowledgement The authors thank Mr. John Cole for his patience in correcting the manuscript.

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